Mechanism Of OVA Nanovaccine With TLR7 Agonist(R848)as Adjuvant In The Treatment Of OVA-induced Allergic Asthma

Objective:By making PLGA-OVA-R848 nano-vaccine,characterizing its morphological characteristics and identifying its encapsulated allergen protein components,the mechanism of the sublingual immunotherapy（SLIT）of chicken ovalbumin（OVA）-induced allergic asthma mouse model was discussed.The ef-ficacy of allergen immunotherapy（AIT）in allergic asthma provides a new strat-egy for the treatment of allergic asthma.Methods:The PLGA-OVA-R848 nano-vaccine was prepared by the dou-ble emulsion solvent evaporation method,and its morphology and particle size were observed by high-resolution scanning electron microscopy;the integrity of OVA protein in the nanoparticles was detected by enzyme-linked immuno-sorbent assay（ELISA）;The antigenicity of proteins in nanoparticles was de-tected by Western blot（WB）;the cumulative natural release rate of nanoparti-cles in vitro was detected by non-interfering protein concentration assay;re-verse transcription real-time quantitative PCR（RT-q PCR）was used to detect the effect of nanoparticles on mouse macrophages（RAW264.7）.Forty BALB/c female mice aged 4-5 weeks were randomly divided into blank control group（n=5）and OVA sensitized group（n=35）.The sensitized group was sensitized by subcutaneous injection of OVA mixed adjuvant AL（OH）₃and challenged by OVA nasal drops,while the normal group was given phosphate buffered saline（PBS）instead;ELISA was used to detect the serum expression of OVA-specific immunoglobulin E（sIgE）in the sensitized group.After the allergic asthma model was successfully established,the mice in the sensitized group were randomly divided into 7 groups and given corresponding drugs for 2 weeks of SLIT,twice a week.After the last treatment,except the normal group mice were given PBS intranasal challenge,the other groups were given 20 ug OVA intrana-sal challenge,once a day for 3 consecutive days.Twenty-four hours after chal-lenge,mice were treated.The serum OVA-specific Ig E,immunoglobulin G1（Ig G1）,and immunoglobulin G2a（Ig G2a）antibody levels of mice in each group were detected by ELISA;leukocytes in the supernatant of bronchoalveolar lav-age fluid and spleen cell culture supernatant were detected by ELISA,as well as interleukin 4,5,13（IL-4,IL-5,IL-13）and Interleukin 10（IL-10）,Interferonγ（IFN-γ）;hematoxylin-eosin staining（H&E）and periodic acid-Schiff（PAS）stain-ing was used to observe the inflammatory infiltration of mouse lung tissue;CCK-8 method was used to detect the inhibition of spleen lymphocyte proliferation in mice;flow cytometry was used to detect the proportion of OVA-specific regula-tory T cells（Tregs）in mouse spleen lymphocytes.Results:PLGA-OVA-R848 nanoparticles are all spherical with smooth sur-face under high-resolution scanning electron microscope,with good appearance,no damage,no cracks,good dispersion of nanoparticles,no adhesion,and their particle size is（219.20±18.56）nm,the encapsulation efficiency was（68.25±2.05）%,and the drug loading was（6.75±0.2）%.The OVA protein in the nanoparticles showed clear bands in SDS-PAGE and WB,respectively,and showed a maximum cumulative release rate of 45.65%after 132 h at 37°C,100rpm/min,p H 7.40.PLGA-OVA-R848 nanoparticles induced increased m RNA expression of interleukin 10（IL-10）and transforming growth factor beta（TGF-β）in RAW264.7.After SLIT,the PLGA-OVA-R848 nanovaccine could effec-tively reduce the levels of sIgE and sIgG1 in the serum of OVA allergic asthma mice,and significantly increase the level of sIgG2a in the serum.PLGA-OVA-R848 nano-vaccine can effectively reduce the contents of IL-4,IL-5 and IL-13cytokines in the bronchoalveolar lavage fluid and spleen cell culture supernatant of allergic asthma mice,and at the same time increase the IL-10 cytokines in the spleen cell supernatant.The PLGA-OVA-R848 nanovaccine can effectively im-prove the hypersecretion of airway mucus in the lung tissue of allergic asthma mice and the infiltration of inflammatory cells around the airway.The PLGA-OVA-R848 nanovaccine can effectively inhibit the proliferation of OVA-specific lymphocytes in the spleen.The PLGA-OVA-R848 nanovaccine can effectively promote the increase in the proportion of OVA-specific Tregs in mouse spleen lymphocytes.Conclusion:In this experiment,a nano-scale allergen vaccine was success-fully prepared,and the allergen protein encapsulated in it can maintain good in-tegrity and antigenicity;The nano-vaccine still has long-acting and sustained-release properties at 37℃and p H 7.40,and can effectively stimulate antigen-presenting cells（APC）to secrete immunoregulatory factors,which provides a good foundation for the immunotherapy of allergic asthma mice;PLGA-OVA-R848 nanovaccine inhibits allergic inflammation by inducing sIgG2a antibody production,preventing sIgE-promoted allergen presentation and specifically binding allergens,and effectively reducing sIgG1 content;The PLGA-OVA-R848 nanovaccine can effectively reverse the polarization of type 2 T helper cells（Th2）in OVA allergic asthma mice,effectively restore the balance between Th2and type 1 T helper cells（Th1）,and improve allergic asthmatic symptoms.PLGA-OVA-R848 nano-vaccine inhibits the excessive proliferation of allergen-specific lymphocytes in the body,and effectively inhibits excessive and strong immune responses;The PLGA-OVA-R848 nanovaccine promotes the prolifera-tion of allergen-specific Tregs and effectively modulates adverse immune re-sponses.